The interaction of nicotine with specific receptors in the brain is known to trigger the release of a number of neurotransmitters, including nitric oxide (NO). While the latter has been established to play an important role in the development of addiction to tobacco products, the underlying molecular pathways remain unclear. Current pharmacological methods for countering the release of NO include inhibition of NO formation and NO scavenging. However, neither one is a viable option for the treatment of nicotine addiction due to the central role endogenous NO plays in cell homeostasis and signaling, and to the deleterious consequences of a systemic reduction in NO availability. Moreover, NO is a constituent of tobacco smoke, and little is known about its actions on downstream effector mechanisms of nicotine. Nicotine metabolism is associated with the formation of redox-active byproducts that can cause oxidative stress through formation of reactive oxygen species (ROS). Because the latter are known to interact strongly with NO, it is likely that nicotine-induced ROS production globally affects the fate and biological actions of NO in the brain and other organs, leading to local nitrosation, nitration and oxidation of biomolecules with profound alterations in protein function and activity. The central hypothesis of this proposal is that local tissue redox poise and nicotine-induced ROS formation are key determinants of the metabolic fate of NO that triggers or facilitates the development of addiction (Stage I). If true, modulation of these determinants may provide a novel targeted approach for suppressing this nicotine-related NO pathway without the adverse effects associated with a global reduction in NO bioavailability (Stage II). To examine this hypothesis, the effects of NO, nicotine and cigarette smoke will be investigated in rodent models of oxidative stress with and w/o antioxidant supplementation or NO pretreatment using a novel metabonomic approach for simultaneous in vivo quantification of free and protein-bound nitroso, nitro and nitrosyl species as well as nitrite and nitrate. In addition, regional changes in cGMP levels, nicotine metabolites and oxidant biomarkers will be measured. Results from these studies are expected to offer an unprecedented insight into the interplay of ROS with NO and important NO-delivery forms in vivo, providing the "building blocks" and conceptual framework for a Stage II application that focuses on novel, targeted intervention strategies in tobacco-related addiction. Tobacco use is a major, worldwide health problem which claims >3 million lives each year. Although the research efforts and public health education in recent years have helped reducing the smoking prevalence in developed countries tobacco use remains the largest single cause of preventable death in the United States and elsewhere. The tragic consequences of nicotine addiction stress the importance of unraveling the molecular mechanisms involved. [unreadable] [unreadable] [unreadable] [unreadable]